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S Cusack

Publications and source records attributed to S Cusack.

At least 19 recordsLinked to original sources
PubMed1998

The crystal structure of asparaginyl-tRNA synthetase from Thermus thermophilus and its complexes with ATP and asparaginyl-adenylate: the mechanism of discrimination between asparagine and aspartic acid.

The crystal structure of Thermus thermophilus asparaginyl-tRNA synthetase has been solved by multiple isomorphous replacement and refined at 2.6 A resolution. This is the last of the three class IIb aminoacyl-tRNA synthetase structures to be determined. As expected from primary sequence comparisons, there are remarkable similarities between the tertiary structures of asparaginyl-tRNA synthetase and aspartyl-tRNA synthetase, and most of the active site residues are identical except for three key differences. The structure at 2.65 A of asparaginyl-tRNA synthetase complexed with a non-hydrolysable analogue of asparaginyl-adenylate permits a detailed explanation of how these three differences allow each enzyme to discriminate between their respective and very similar amino acid substrates, asparagine and aspartic acid. In addition, a structure of the complex of asparaginyl-tRNA synthetase with ATP shows exactly the same configuration of three divalent cations as previously observed in the seryl-tRNA synthetase-ATP complex, showing that this a general feature of class II synthetases. The structural similarity of asparaginyl- and aspartyl-tRNA synthetases as well as that of both enzymes to the ammonia-dependent asparagine synthetase suggests that these three enzymes have evolved relatively recently from a common ancestor.

Adenosine Triphosphate↗
PubMed1998

tRNA(Pro) anticodon recognition by Thermus thermophilus prolyl-tRNA synthetase.

BACKGROUND: Most aminoacyl-tRNA synthetases (aaRSs) specifically recognize all or part of the anticodon triplet of nucleotides of their cognate tRNAs. Class IIa and class IIb aaRSs possess structurally distinct tRNA anticodon-binding domains. The class IIb enzymes (LysRS, AspRS and AsnRS) have an N-terminal beta-barrel domain (OB-fold); the interactions of this domain with the anticodon stem-loop are structurally well characterised for AspRS and LysRS. Four out of five class IIa enzymes (ProRS, ThrRS, HisRS and GlyRS, but not SerRS) have a C-terminal anticodon-binding domain with an alpha/beta fold, not yet found in any other protein. The mode of RNA binding by this domain is hitherto unknown as is the rationale, if any, behind classification of anticodon-binding domains for different aaRSs. RESULTS: The crystal structure of Thermus thermophilus prolyl-tRNA synthetase (ProRSTT) in complex with tRNA(Pro) has been determined at 3.5 A resolution by molecular replacement using the native enzyme structure. One tRNA molecule, of which only the lower two-thirds is well ordered, is found bound to the synthetase dimer. The C-terminal anticodon-binding domain binds to the anticodon stem-loop from the major groove side. Binding to tRNA by ProRSTT is reminiscent of the interaction of class IIb enzymes with cognate tRNAs, but only three of the anticodon-loop bases become splayed out (bases 35-37) rather than five (bases 33-37) in the case of class IIb enzymes. The two anticodon bases conserved in all tRNA(Pro), G35 and G36, are specifically recognised by ProRSTT. CONCLUSIONS: For the synthetases possessing the class IIa anticodon-binding domain (ProRS, ThrRS and GlyRS, with the exception of HisRS), the two anticodon bases 35 and 36 are sufficient to uniquely identify the cognate tRNA (GG for proline, GU for threonine, CC for glycine), because these amino acids occupy full codon groups. The structure of ProRSTT in complex with its cognate tRNA shows that these two bases specifically interact with the enzyme, whereas base 34, which can be any base, is stacked under base 33 and makes no interactions with the synthetase. This is in agreement with biochemical experiments which identify bases 35 and 36 as major tRNA identity elements. In contrast, class IIb synthetases (AspRS, AsnRS and LysRS) have a distinct anticodon-binding domain that specifically recognises all three anticodon bases. This again correlates with the requirements of the genetic code for cognate tRNA identification, as the class IIb amino acids occupy half codon groups.

Amino Acid Sequence↗
PubMed1997

The crystal structure of the signal recognition particle Alu RNA binding heterodimer, SRP9/14.

The mammalian signal recognition particle (SRP) is an 11S cytoplasmic ribonucleoprotein that plays an essential role in protein sorting. SRP recognizes the signal sequence of the nascent polypeptide chain emerging from the ribosome, and targets the ribosome-nascent chain-SRP complex to the rough endoplasmic reticulum. The SRP consists of six polypeptides (SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72) and a single 300 nucleotide RNA molecule. SRP9 and SRP14 proteins form a heterodimer that binds to the Alu domain of SRP RNA which is responsible for translation arrest. We report the first crystal structure of a mammalian SRP protein, that of the mouse SRP9/14 heterodimer, determined at 2.5 A resolution. SRP9 and SRP14 are found to be structurally homologous, containing the same alpha-beta-beta-beta-alpha fold. This we designate the Alu binding module (Alu bm), an additional member of the family of small alpha/beta RNA binding domains. The heterodimer has pseudo 2-fold symmetry and is saddle like, comprising a strongly curved six-stranded amphipathic beta-sheet with the four helices packed on the convex side and the exposed concave surface being lined with positively charged residues.

Amino Acid Sequence↗
PubMed1997

Crystal structure analysis of the activation of histidine by Thermus thermophilus histidyl-tRNA synthetase.

The crystal structure at 2.7 A resolution of histidyl-tRNA synthetase (HisRS) from Thermus thermophilus in complex with its amino acid substrate histidine has been determined. In the crystal asymmetric unit there are two homodimers, each subunit containing 421 amino acid residues. Each monomer of the enzyme consists of three domains: (1) an N-terminal catalytic domain containing a six-stranded antiparallel beta-sheet and the three motifs common to all class II aminoacyl-tRNA synthetases, (2) a 90-residue C-terminal alpha/beta domain which is common to most class IIa synthetases and is probably involved in recognizing the anticodon stem-loop of tRNA(His), and (3) a HisRS-specific alpha-helical domain inserted into the catalytic domain, between motifs II and III. The position of the insertion domain above the catalytic site suggests that it could clamp onto the acceptor stem of the tRNA during aminoacylation. Two HisRS-specific peptides, 259-RGLDYY and 285-GGRYDG, are intimately involved in forming the binding site for the histidine, a molecule of which is found in the active site of each monomer. The structure of HisRS in complex with histidyl adenylate, produced enzymatically in the crystal, has been determined at 3.2 A resolution. This structure shows that the HisRS-specific Arg-259 interacts directly with the alpha-phosphate of the adenylate on the opposite side to the usual conserved motif 2 arginine. Arg-259 thus substitutes for the divalent cation observed in seryl-tRNA synthetase and plays a crucial catalytic role in the mechanism of histidine activation.

Amino Acid Sequence↗
PubMed1997

Aminoacyl-tRNA synthetases.

Crystallographic studies of a number of aminoacyl-tRNA synthetases and their complexes with ATP, amino acid and cognate tRNA are leading to an increasingly detailed picture of how these sophisticated enzymes function. Within the two distinct structural classes of ten synthetases, many common features are apparent, although evolution has led to many interesting idiosyncrasies in certain enzymes. Recent advances, specifically concerning class II enzymes, have increased our knowledge of both the role of electrophiles in the mechanism of amino acid activation and cross-subunit tRNA recognition and help solve the evolutionary puzzles that have emerged from the extension of the aminoacyl-tRNA synthetase database to include Archae.

Adenosine Triphosphate↗
PubMed1997

Paramedics, technicians, and survival from out of hospital cardiac arrest.

OBJECTIVE: To test the hypothesis that limited paramedic advanced life support skills afford no advantage in survival from cardiac arrest when compared with non-paramedic ambulance crews equipped with defibrillators in an urban environment; and to investigate whether separate response units delayed on scene times. METHODS: A prospective, observational study was conducted over 17 consecutive months on all adult patients brought to the accident and emergency (A&E) department of Glasgow Royal Infirmary having suffered an out of hospital cardiac arrest of cardiac aetiology. The main interventions were bystander cardiopulmonary resuscitation (CPR) and limited advance life support skills. MAIN OUTCOME MEASURES: Return of spontaneous circulation, survival to admission, and discharge. RESULTS: Of 240 patients brought to the A&E department, 19 had no clear record of whether a paramedic was or was not involved and so were excluded. There was no difference in survival between the two groups, although a trend to admission favoured non-paramedics. Paramedics spent much longer at the scene (P < 0.0001). Witnessed arrests (P = 0.01), early bystander CPR (P = 0.12), shockable rhythms (P = 0.003), and defibrillation (P < 0.0001) were associated with better survival. Intubation and at scene times were not associated with better survival. Delayed second response units did not prolong at scene times. CONCLUSIONS: The interventions of greatest benefit in out of hospital cardiac arrest are basic life support and defibrillation. Additional skills are of questionable benefit and may detract from those of greatest benefit.

Adolescent↗
PubMed1997

Mutational analysis of the protein subunits of the signal recognition particle Alu-domain.

Two polypeptides of the murine signal recognition particle (SRP), SRP9 and SRP14, bind exclusively as a heterodimer to SRP RNA and their presence is required for elongation arrest activity of the particle. SRP9/14 also constitute a subunit of small cytoplasmic Alu RNPs. To identify RNA-binding determinants, we assayed the dimerization and RNA-binding capacities of altered proteins in vitro. Despite the structural homology of the two proteins, their requirements for dimerization differ substantially. In SRP9, an internal fragment of 43 amino acids is sufficient to allow dimer formation, whereas in SRP14 only few changes, such as removing an internal loop region, are tolerated without affecting its dimerization activity. The dimerization defect of the SRP14 proteins is most likely explained by a reduced stability or ability to fold of the proteins. Interestingly, SRP RNA can engage certain dimerization-defective SRP14 proteins into stable complexes, suggesting that low-affinity interactions between the RNA and SRP14 may help to overcome the folding defect or the reduced stability of the proteins. We identified two regions, one in each protein, that are essential for RNA-binding. In SRP9, acidic amino acid residues in the N-terminal alpha-helix and the adjacent loop and, in SRP14, a flexible internal loop region are critical for RNA-binding. In the heterodimer, the two regions are located in close proximity, consistent with the RNA-binding region being formed by both proteins.

Amino Acid Sequence↗
PubMed1997

Identification of a minimal Alu RNA folding domain that specifically binds SRP9/14.

We have identified functionally and analyzed a minimal Alu RNA folding domain that is recognized by SRPphi14-9. Recombinant SRPphi14-9 is a fusion protein containing on a single polypeptide chain the sequences of both the SRP14 and SRP9 proteins that are part of the Alu domain of the signal recognition particle (SRP). SRPphi14-9 has been shown to bind to the 7SL RNA of SRP and it confers elongation arrest activity to reconstituted SRP in vitro. Alu RNA variants with homogeneous 3' ends were produced in vitro using ribozyme technology and tested for specific SRPphi14-9 binding in a quantitative equilibrium competition assay. This enabled identification of an Alu RNA of 86 nt (SA86) that competes efficiently with 7SL RNA for SRPphi14-9 binding, whereas smaller RNAs did not. The secondary structure of SA86 includes two stem-loops that are connected by a highly conserved bulge and, in addition, a part of the central adaptor stem that contains the sequence at the very 3' end of 7SL RNA. Circularly permuted variants of SA86 competed only if the 5' and 3' ends were joined with an extended linker of four nucleotides. SA86 can thus be defined as an autonomous RNA folding unit that does not require its 5' and 3' ends for folding or for specific recognition by SRPphi14-9. These results suggest that Alu RNA identity is determined by a characteristic tertiary structure, which might consist of two flexibly linked domains.

Animals↗
PubMed1996

The crystal structures of T. thermophilus lysyl-tRNA synthetase complexed with E. coli tRNA(Lys) and a T. thermophilus tRNA(Lys) transcript: anticodon recognition and conformational changes upon binding of a lysyl-adenylate analogue.

The crystal structures of Thermus thermophilus lysyl-tRNA synthetase, a class IIb aminoacyl-tRNA synthetase, complexed with Escherchia coli tRNA(Lys)(mnm5 s2UUU) at 2.75 A resolution and with a T. thermophilus tRNA(Lys)(CUU) transcript at 2.9 A resolution are described. In both complexes only the tRNA anticodon stem-loop is well ordered. The mode of binding of the anticodon stem-loop to the N-terminal beta-barrel domain is similar to that previously found for the homologous class IIb aspartyl-tRNA synthetase-tRNA(Asp) complex except in the region of the wobble base 34 where either mnm5 s2U or C can be accommodated. The specific recognition of the other anticodon bases, U-35 and U-36, which are both major identity elements in the lysine system, is also described. Additional crystallographic data on a ternary complex with a lysyl-adenylate analogue show that binding of the intermediate induces significant conformational changes in the vicinity of the active site of the enzyme.

Amino Acid Sequence↗
PubMed1996

The crystal structure of the ternary complex of T.thermophilus seryl-tRNA synthetase with tRNA(Ser) and a seryl-adenylate analogue reveals a conformational switch in the active site.

The low temperature crystal structure of the ternary complex of Thermus thermophilus seryl-tRNA synthetase with tRNA(Ser) (GGA) and a non-hydrolysable seryl-adenylate analogue has been refined at 2.7 angstrom resolution. The analogue is found in both active sites of the synthetase dimer but there is only one tRNA bound across the two subunits. The motif 2 loop of the active site into which the single tRNA enters interacts within the major groove of the acceptor stem. In particular, a novel ring-ring interaction between Phe262 on the extremity of this loop and the edges of bases U68 and C69 explains the conservation of pyrimidine bases at these positions in serine isoaccepting tRNAs. This active site takes on a significantly different ordered conformation from that observed in the other subunit, which lacks tRNA. Upon tRNA binding, a number of active site residues previously found interacting with the ATP or adenylate now switch to participate in tRNA recognition. These results shed further light on the structural dynamics of the overall aminoacylation reaction in class II synthetases by revealing a mechanism which may promote an ordered passage through the activation and transfer steps.

Amino Acid Sequence↗
PubMed1996

Crystallization and preliminary crystallographic analysis of the signal recognition particle SRPphi14-9 fusion protein.

The SRPphi14-9 fusion protein, which can functionally replace the SRP9/14 heterodimer in the mammalian signal recognition particle (SRP), has been crystallized using the vapor diffusion method. Four different crystal forms were grown. SRPphi14-9 form IV crystals belong to the space group P4(1)22/ P4(3)22 with cell parameters a = b = 69.7 Angstroms, c = 95.7 Angstroms, alpha = beta = gamma = 90 degrees. A complete data set to 2.8 Angstroms resolution with an Rsym on intensities of 7.0% was collected on a single flash-frozen crystal.

Amino Acid Sequence↗
PubMed1996

Crystallization and preliminary X-ray analysis of the 9 kDa protein of the mouse signal recognition particle and the selenomethionyl-SRP9.

Two different crystal forms of the 9 kDa protein of the signal recognition particle (SRP9) have been prepared by the hanging drop vapor diffusion technique using 28% (w/v) PEG8000 or 28% saturated ammonium sulphate as precipitant. The crystals are hexagonal bipyramids with average dimensions of 0.2 X 0.1 X 0.1 mm(3) and they diffract to a resolution of 2.3 Angstroms. They belong to the space groups P6(2)22/P6(4)22 or P3(1)21/P3(2)21 with cell dimensions a = b = 63.0 Angstroms, and c = 111.5 Angstroms. Crystals have also been grown from the selenomethionyl protein and multiwavelength data sets have been collected.

Animals↗
PubMed1996

The structure of the Escherichia coli EF-Tu.EF-Ts complex at 2.5 A resolution.

The crystal structure of the EF-Tu.EF-Ts complex from Escherichia coli has been determined to a resolution of 2.5 A. The complex contains two subunits of each of the elongation factors. The two EF-Ts molecules form a tight dimer, but there is little contact between the two EF-Tu molecules. The interaction of EF-Ts with EF-Tu results principally in the disruption of the Mg2+ ion binding site, thereby reducing the affinity of EF-Tu for guanine nucleotides.

Amino Acid Sequence↗
PubMed1995

The structural basis for seryl-adenylate and Ap4A synthesis by seryl-tRNA synthetase.

BACKGROUND: Seryl-tRNA synthetase is a homodimeric class II aminoacyl-tRNA synthetase that specifically charges cognate tRNAs with serine. In the first step of this two-step reaction, Mg.ATP and serine react to form the activated intermediate, seryl-adenylate. The serine is subsequently transferred to the 3'-end of the tRNA. In common with most other aminoacyl-tRNA synthetases, seryl-tRNA synthetase is capable of synthesizing diadenosine tetraphosphate (Ap4A) from the enzyme-bound adenylate intermediate and a second molecule of ATP. Understanding the structural basis for the substrate specificity and the catalytic mechanism of aminoacyl-tRNA synthetases is of considerable general interest because of the fundamental importance of these enzymes to protein biosynthesis in all living cells. RESULTS: Crystal structures of three complexes of seryl-tRNA synthetase from Thermus thermophilus are described. The first complex is of the enzyme with ATP and Mn2+. The ATP is found in an unusual bent conformation, stabilized by interactions with conserved arginines and three manganese ions. The second complex contains seryl-adenylate in the active site, enzymatically produced in the crystal after soaking with ATP, serine and Mn2+. The third complex is between the enzyme, Ap4A and Mn2+. All three structures exhibit a common Mn2+ site in which the cation is coordinated by two active-site residues in addition to the alpha-phosphate group from the bound ligands. CONCLUSIONS: Superposition of these structures allows a common reaction mechanism for seryl-adenylate and Ap4A formation to be proposed. The bent conformation of the ATP and the position of the serine are consistent with nucleophilic attack of the serine carboxyl group on the alpha-phosphate by an in-line displacement mechanism leading to the release of the inorganic pyrophosphate. A second ATP molecule can bind with its gamma-phosphate group in the same position as the beta-phosphate of the original ATP. This can attack the seryl-adenylate with the formation of Ap4A by an identical in-line mechanism in the reverse direction. The divalent cation is essential for both reactions and may be directly involved in stabilizing the transition state.

Adenosine Monophosphate↗
PubMed1995

Cocrystallization of lysyl-tRNA synthetase from Thermus thermophilus with its cognate tRNAlys and with Escherichia coli tRNAlys.

Lysyl-tRNA synthetase from Thermus thermophilus has been cocrystallized with either its cognate tRNAlys or Escherichia coli tRNAlys using ammonium sulfate as precipitant. The crystals grow from solutions containing a 1:2.5 stoichiometry of synthetase dimer to tRNA in 18-22% ammonium sulfate in 50 mM Tris-maleate buffer at pH 7.5. Both complexes form square prismatic, tetragonal crystals with very similar unit cell parameters (a = b = 233 A, c = 119 A) and diffract to at least 2.7 A resolution. However the homocomplex is of space group P42(1)2 and the heterocomplex of space group I422.

Crystallization↗
PubMed1995

Crystallization of Thermus thermophilus histidyl-tRNA synthetase and its complex with tRNAHis.

Histidyl-tRNA synthetase (HisRS) has been purified from the extreme thermophile Thermus thermophilus. The protein has been crystallized separately with histidine and with its cognate tRNAHis. Both crystals have been obtained using the vapor diffusion method with ammonium sulphate as precipitant. The crystals of HisRS with histidine belong to the spacegroup P2(1)2(1)2 with cell parameters a = 171.3 A, b = 214.7 A, c = 49.3 A, alpha = beta = gamma = 90 degrees. A complete data set to a resolution of 2.7A with an Rmerge on intensities of 4.1% has been collected on a single frozen crystal. A partial data set collected on a crystal of HisRS in complex with tRNAHis shows that the crystals are tetragonal with cell parameters a = b = 232 A, c = 559 A, alpha = beta = gamma = 90 degrees and diffract to about 4.5 A resolution.

Crystallography, X-Ray↗